PROJECTSUMMARY Theincidenceofacuteandchronicwoundsfromsurgery,traumaticinjury,diabetes,andmetabolicsyndromeis on the rise, affecting over three million people in the US alone and costing tens of billions of dollars a year. Successfulwoundhealingprogressesthroughcoagulation/inflammation,proliferation,andremodelingphases, culminating in tissue regeneration and/or contraction and scarring. Macrophages are essential mediators of progression through these stages, and responsive to external therapeutic modulation. Yet, the soluble and mechanicalfactorsmediatingmacrophageinteractionswiththesematerialsandotherwoundeffectorsremain largelyunknown.Iproposetouseimmunologyandbioengineeringapproachestoimproveourtoolsandworkto fill this unmet clinical need. Our previous work demonstrated that fibrin, a major component of the provisional extracellular matrix, skews murine bone marrow derived macrophages (BMDM) towards an anti-inflammatory phenotypeinvitro,suppressingthestereotypedcytokineresponsetopotentinflammatorystimuli,includingLPS. Preliminary data from 5 mm full-thickness skin wounds in mice showed anti-inflammatory skew in the wound, measuredbyArginase,iNOS,andYAPexpressioninmacrophages,anddiminishedmyofibroblastmarkeralpha smoothmuscleactin(aSMA)withfibrintreatment,suggestingaroleformacrophage-myofibroblastinteractions inthewound.Additionally,bothsoft(1.2kPa)andstiff(840kPa)gelshavebeenshowntoincreasemacrophage secretionofinflammatorycytokinesseeninthestereotypedforeignbodyresponse(e.g.TNFa),withintermediate stiffness showing lower secretion, indicating a mechanical sweet spot for immunomodulation. I propose to engineerfibrin-PEGhydrogels,mechanicallyoptimizedformacrophageimmunomodulatorycapacity,andapply themtoinvitrococulturestodeterminemacrophage-fibroblast-fibrininteractiondynamicsandtestthehypothesis that fibrin hydrogels of moderate stiffness (~140 kPa) will best promote anti-inflammatory macrophage phenotype,facilitatingdecreasedmyofibroblastcontractility.Ialsoaimtodefinethemechanismsbywhichfibrin hydrogels modulate macrophage phenotype to achieve wound healing and regeneration in vivo, using a full thickness skin wound model in wild type, and selective immune cell depletions models (macrophage- MAFIA, clodrosome;?neutrophil-anti-GR1,B-cell-anti-CXCL13).Iwillusehistology,flowcytometry,andRNA-seqtotest thehypothesisthatfibrin-PEGhydrogelsofmoderatestiffness(~140kPa)willacceleratewoundresolutionand tissue regeneration by promoting anti-inflammatory macrophage phenotype. Understanding the mediators of fibrin-enhanced wound healing will help us better understand physiologic wound repair, and will lay the groundworkforrationaldesignofnoveltherapeutics,andbetterwoundcareforpatients.